,s c-qct 3

Ratios of biogenic elements for distinguishing recent from fossilmicroorganisms

Richard B. Hoover

Astrobioiogy Laboratory,

NASA/Marshall Space Flight Center,

National Space Science and Technology Center,

320 Sparkman Dr., Htmtsville, AL 35805 USA

ABSTRACT

The ability to distmgnish possible mierofossils from recent biolo_eal contm_nants is of great importance to

Astrobiology. In fftis paper we discuss the application of the ratios of life critical biogeme elements (C/O; C/N; andC/S) as determined by Energy Dispersive X-ray Spectroscopy (EDS) to this problem. Biognnic element ratios will beprovided for a wide variety of living ¢yanobaeteria and other microbial extremophiles, preserved herbarium materials,and ancient biota from the Antarctic Ice Cores and SibetSan and Alaskan Permafrost for comparison with megafossilsand mierofossil in ancient terrestrial rooks and carbenace_us meteorites.

KEYWORD$: M.ierofossils, Carbonaceous Meteo_tes, Orgueil, Murehison, Energy Dispersive X-Ray Spectroscopy;

Biomarkerso Biogenie Elements, Elemental Ratios, Cyanobaeteria, Extremophiles

L _TI/,ODUCTION

Astrobiologists have highlighted the importmme of establishing the validity of ehemie_, mineralogical andmorphological biomarkers in anoient roekso meteorites and other Astromaterials. It is neeessm3J to recognize thatbiomarkers exist in a variety of strength levets. Weak biomarkers only suggest biogenicity whereas strong, validbiomarkers provide clear evidence of biological activity. Valid biomarkers in ancient rocks, meteorites and other

astromateriats must satisfy rigorous criteria, During the past decade, extensive Field Emission 0VESEM) andEnvironmental (ESE/VD Scanning Electron Microscopy investigations of the morphological characteristics and EDSanalyses of the elemental compositions of minerals, ten'estrial microfossits and known biological organisms have beeninvestigated in the NAS_SFC/NSSTC. Astrobiology Laboratory. These studies mctuded a wide variety ofminerals, rocks; and meteorites as well as living and fossil (Holooene to Arehaean 2.80a) eukaryotic and prokaryotie

orgamsms (e.g., plants, hair, fungi, diatoms and other algae, cyanobacteria, sulfur and sulfate reduoing bacteria. Thisresearch has resulted in the recognition that elemental ratios of certain life-critical biogenie elements can provide a

powerfifl meohanism for distinguishing r_eent biologioat contaminants from aneiem indigenous mJcrofossils andreeogn'iz..ing valid morphological biomarkers in meteorites. This paper addresses the use of ratios of life crkieal

biogenic elements (C, N, and S) for distinguishing recent biological comaminants from valid, indigenous mierofossilsin ancient rocks and meteorites, To be considered valid, the biomarkers must_ satisfy to rigorous criteria:

1,) Vaikl bt_markers mt_t be Unambiguously Biologist!

2.) Valid biomarkers mastbe Undeniably Indigenous

Althottgh many biominerals provide weak evidenoe of biogeni_ity, some can be considered strongbiomarkers. These include conclusively recognizable biofikns; biogenic magnetites and magnetosomes in "chain ofpearls" configurations. Other mineral, biomarkers inelude silica biopotymers such as are found Lr_the shell of diatoms,silieoflagellates, and radiolar_ More definitive evidence of biologieal activity is provided by valid biomarkers such

as complex i_prenoid biomoleeule_, fatty aoids, cholest_me Pristane, Phytane and the diagenetie breakdown products

https://ntrs.nasa.gov/search.jsp?R=20070037446 2020-01-26T16:17:28+00:00Z

ofother biological pigments and protein Amino Acids with lmambiguous enantiomeric excess. Valid biomarkers alsowould include segments of RNA. DNA. genes, protems_ enzymes_ or other complex biomoleeutes as well andtmambiguously recognizable mierofossils or consortia with distinctive chemical and cellular differentiation. It iscrucial that the mdigeneity of the biomarkers can be undem'ably established. For this reason methodologies must be

developed to distinguish recent biological contaminants from valid indigenous ehemicat and morphologicalbiomarkers and mierofossfls in ancient reeks, meteorites and retume, d astromaterials.

Many important ehe_nical biomarkers trove been detected in oarbona_eous meteorites during studies carried

out by different investigators on a carbonaceous meteorite sables studied from the late-1800' s to the present. Table Iis a chronological stmmu_y for many chemical biomarkers detected in carbonaceous meteorites.

TABLE I. Chemical Biomarkers in, Carbonaceous Meteorites

BIOMARKER

Long-Chain Fatty

Porpbyrlns _a°

....... REFERENCE

Cloez, 1864a, 1864b; Pisani, 1864

Nagy etaL, t961; Kvenvolden etal., I970

and Bit.z, 1963

and Nagy, 1966

Commins .and Ha_ington, 1966

Some of these ehemieat biomarkers (e.g PAH's_ Petroleum-like Hydropearbons, Amino Acids, etc.) can beproduced by abiofie mechanisms (e.g., Miller-Urey or Fisher-Tmpseh _nthesis) and they consequently do not provide

conclusive evidence of biological activity, However, many of the others are not produced by any known abiofiemechanism:s aad thus must be considered to represent valid chemical biomarkers in carbonaceous meteorites.

In 1967, Nooner and O1"613 detected isoprenoids (Pristane, Phytane and Norpristane) in the Orgueil CI1carbonaceous meteorite_ Pristane (2,6,10,t4-tetramethylpentadecane) is a _aturally saturated terp_mpoid alkane(Cl_-h0) that is a biogeoehemical derivative from phytot. Phytol is a well-known a decomposition product of

Chlorophyll. This photosy, nLhefie pigment, which is crucial for the complex proce_ of photosynthesis, ts common mphototrophie organisms on Earth. It plays a critical role in the flow of energy in the Biosphere_ Phototrophs usetrapped solar energy to cortvert, carbon dioxide and water into sut_ars and more complex eetlular components such ascarbohydrates_ polysaeeharide sheaths, fatty acids, nueleie acids, DNA, RNA and a host of other bioehemicalsessential for .t{fe. The organic carbon that is fixed-from carbon dioxide can then be used by other life forms. The storedsolar energy _s extracted by eh_,nno_oplfic organisms via anaerobic fermentation processes that occur in the ab_nee ofoxygen, During aerobic _spimtion the organic carbon is completely oxidized and returned to the atmosphere ascarbon dioxide, thereby completing the carbon cycle. Every carbon atom in every molec_fle inthe organism _s derivedfrom carbon dioxide that is f_ed into organic carbon by the photch_ynthefic process.

There are no knowrt abiofie mechanisms that produce Chlorophyll, Pristane and Phfiane are diagenefiederivative breakdown products of chlorophyl1 and are considered valid biomarkers. Kissinm has pointed out that thetransformation of ekloropbyll into its phytol chain and then to the isoprenoid atkanes pristine and phytane is a slow

process. Phytane O,7,11,t 5-tetramethylhexadecane) is only found on F_rth m ancient oil shales and eeade petroleum.The conversion of terrestrial biological matter into linear alkyl ehairts _ a slow, multi-step process involvingmicrobialy mediated _mzymatic reactions. Kissm's experimental investigations establishe_J that the free saturated

hydrocarbons in the Orgueit meteorite were not the result of contamination by oits used in the laboratory environmentor recent mierobioloeical contamination effects.

1. Biogenic Elemeats ht lJviag Orgaaisms and Meteorites

All orgamsms on Ea_ appear to possess the same fum_znental requirements for liquid water, an energysource and requirement for a limited set of life-critical (biogenic) elements. By far the most critical elements in life on

Earth are file six life critical elements (Carbon, Hydrogen, Oxygen, Nitrogen, Phosphorus, and Sulfur) are found inrelatively large quantities in all.organisms, The first four are by far the most abuadmat, Sdflar and Phosphorus are alsoerificat for life, bUt they are found in much smaller quantities in living organisms, Minor biogenie elements present inorganisms in lesser amounts include: Mg_ Fe, Na, K, Ca_ and Ct, Biogenie elements are needed for constriction of

cellular structural components_ metabolism and respiration, and storage and transport of energy mad informotion. Afew other elements (e,g, Si, Mla, A1, I, Cu, Zn, As_ Ni and F) are needed for enzymatic actions and specializedfunctions and they usually appear only in trace levels.

All of the major life-critical bi0genie elements (along with Fe, Si, and Mg) are also the major components ofboth earbonac, eous meteorites and comets. Comets may have played a major rote in the dispersal of the biogenicelements throt_oUt the Solar System, 3Q'nAll are found in the Orgueil, Murchdson and other carbonaceous meteorites.

The distributions of the major biogenic elements within the meteorite samples are very heterogeneous, EnergyDispersive X-Ray Spectroscopy (EDS) spot analysis and 2-D X-ray maps obtained during the present, investigationclearly establish that the biogenic dements C, O, Mg, and S are heavily eoneentrat_ in the filaments found in theOrgueil CI1 carbonaceous meteorite,. Furthermore, there is a readily observable differentiation in abandances of the

bingenic elements from observably different and distinguishable conknonents of the filaments,Nitrogen is ve_ rarely found to be present in the f_kaments above the level of detectability of the EDS.

Nitrogen is absolutely essential for life since it is presem in all mnine acids and in the pufines (Adenine and Guanine)mad Pyrimidinos (Cytosine, Uracil, and Thymine) which are essential for the construction of life critical biomoleedes

such as ATP, RNA, DNA and proteins. All modem (and old but not fossilized) biological materials studied have beenfound to have detectable bevels of nitrogen. However; nitrogen is absent in many ancient fossils as a result of thediagenetie losses that oeeu_ over many millions of years.

1.1 Carbon in carbonaceous meteorites attd filamentous microfossils

Mason _a reported in 1963-that carbonaceous meteor-ires can contain up to 4.8% (weight %) carbon, erringand Zahringer u obtained a slightly lower "value (3,19 wt %), Using the more sensitive Energy Loss Electron

Spectrometer (EELS), the bulk Carbon content of the Orgueil meteorite was found to be 3.5% (by weight) and theyfound 5% of the carbon rich _s to also be enriched in Oxygen, Phos-phoms and Sulfur (the COPS phase). _ The

Biosphere is critically dependent upon "carbon fixation" reactions. In tlfis complex processes enzymes are used to

catalytically eonvert atmospheric-CO2 into carbohydrates that can be used by living organisms. During the carbonfixation eycte, light energy is captured during photosynthesis by photoautotrophi¢ microorganisms and stored in thechemical bonds of ATP atld NADP. This stored energy can then be used to power the enzymatic reactions _t convertmolecules of carbon dioxide and water into the orgmlic mole0ules. During the Calvin cycle in living organisms thefixation of CO_ into carbohydrates is catalyzed by the (RuBisC_) enzymef _ The RuBisCo enzyme has both

earboxytase and oxygenase activity and is also capable of fixing atmospheric oxygen during the process ofphotorespiration. Rtfl3isCo comprises Mm0st 50% of the protein of chloroplasts and is thought to be the- mostabundant protein on Earth. M0axy bacteria and arctaaea are _hemoautotrophs or chenlolithotrophs that are capable ofderiving energy from inorganic energy sources and chemical reactions and synthesizing organic compounds fromcarbon dioxide or carbonate rocks. Biologi¢al fractionation occurs during the Calvin cycle since the light carbon s_ableksotoi m (C12) is incoffJomted in preference to the hea_er (C _) isotope. Carbon iset0pe measurements for glycme mthe Orgucil meteorite yielded a value of 51"_C_+22 per nail (far above the terrestriM range of -20 to -35 per miIproviding clear and convincing evidence that the Orgueil amino acids are extraterrestrial. _ Biological fraetionafion of

stable isotopes of carbon as seen m ancient long chain carbon biogeopolymers (kerogen) has been interpreted asgeological evidence of biological activity preserved in the rock record for _3.8 Ga. Stromatolites formed byfilamentous prokaryotes and cyanobaeteria extend back to at least 2.8 Gas741

1.2 Oxygen in fire earbonacemls meteoriten and fllamen*ous mlcr_fossils

Oxygen is a life-critical biogeme component of virmatly alt organic molecules, RNA, DNA and proteins and

it is erueiaI for metabolism, The Orgueil and Murehison meteorites are rich in oxygen. The mean oxygen content for

CImeteoritesis46%fa The EDS spot analysis reveals that some of the filaments have .Oxygen levels above 60%

(atomic) while others have values as low as 9% atomio, Some ofthe sheaths have C/O ratios similar to kerogen. Forexample CK) = 108 at spot X on the sheath of the graphitized filamentous mierofossil in Fig. 2_a, This ratio is entirelyunlike that obtained for living or recently 4cad biological matter, Elemental 2-D x-ray maps show many of the other

filaments often have oxygen levels that si_gnifieanfly exceed the levels found in the adjacent or underlying reek matrix.

1.3 Nitrogen _t the Biosphere

The Biosphere is critically dependent upon both "carbon fixation" and "nitrogen fixation" reactions. In thesecomplex processes enzymes are used to catalytically convert atmospheric CO2 ,and N2 into forms that are useful toliving organisms. Nitrogen is one of the most abundant elements in the Solar System and it critical to all livingorganisms, Nitrogen is an esmntiat component of amino acids, nucleic acids, purines, pyrimidines, ATP and proteins.

Nitrogen is required for syrahesis of RIgA and DNA and the regulation of many crucial biochemical pathways.Although nitrogen is atrandant m the atmosphere (_)%), atmospheric nitrogen emmet be use_ by livi_r_g orgamsmsuntil it has been converted by "nitrogen fLxation" enzTcmatie pro_sses. The N2 gas molecule is unavailable for most

organisms due to the triple bond that renders .it virtually inert. Nitrogen is converted into a useable state by nitrogenfixing bacteria such as the .heteroeystous eyanobaeteria. The process of nitrogen fixation involves the conversion ofgaseous dinitrogen into ammorda Nl:h*, whi0h 0an then be com_erted to nitrite or nitrate ions by nitrifying bacteria.Nitrogen fixation is accomplished by use of the enzyme complex nitrogenase which catalyzes the reaction:

N2+ 8 e- +gFY + 16 MgATP ") 2NHs +H2 + 16MgATP + t6 Pi

Fixed nitrogen is precious and is scavenged after death by microorganisms and subsequently removed by avery slow diagenetie proees_s. Although the level of nitrogen earl be fairly high (2 to t5% in living and dead modembiological materials, ,it is almost never enootmtered at levels above 2% in miorofossils. Consequently, nitrogen levels

and C/N ratios provide a use_ tool for distmguishh!g recent biological eontaminams fl'om indigenous mierofossils.Cyanobaeteria play a crucial role in nitrogen fixation on planet F,arth. The nitrogen content of living

eyanobaeteria often amount to more than 10% by weight *_A nitrogen deficiency immediately affects the amotmt ofphyeobiliproteias and eonsequent!y their photosynthetic light harvesting efficiency, The nitrogenase enzymes are

extremely sensitive to oxygen and therefore in order to fix nitrogen an anaerobic environment must be provided.Cyanobaeteria are oxyg_fic pt_otoautotrophie mieroorganis-a_s that are primarily aerobic microorganisms andtherefore they must provide specialized oMls to faoititate this prooess. The diazotroptfie eyanohaeteria (capable of

using N2 as their s01e souroe of nitrogen for growth) earl be subdivided into three grot_ps:Group I: Heterocystous Cytmobacteria: Exclusively filamentous eyanobaeteria that differentiate special cells

(heteroeysts) which have lost the eapaei .ty for oxygenic photo_nthesis. Thick walled heteroeysts form a diffusionbarrier for gases mad limit the entry of oxygen so that they can carry out diazotrophie growth 'trader fully aerobic

conditions. The heterocyst prevents oxygen from entering the cell where the nitrogen fixation is taking place.Heteroeystous 0yanobaoteria include: Calothrix, Anabaena, Nodutaria, and "Scytonema. The Orgueil meteoriteoontains filaments interpreted as morphotypes of Calothrix, but the other Genera have yet been deteoted.

Group 11: Anaerobic N_--Fixing Nott-tteterocy_tous Cyanobacterla: These are bc_th l_flementous anduni_ellular. They ftmetion by locating themselves in miorobial mats in regimes that allow them to avold oxygen andthey may require sulfide to inhibit oxygenic photosynthesis. Exmnples inolude: Oscil#_toria limnetica, Ptectonemaboryanum, and several species of Lyngbya and Syneehococcus. Most of the filamentous forms found in Orgueil areconsistent with morphotypes of species of Oscillatoeia and PIectonema_

Group tH: Aerobic N-z-Fixing Non-Heterocystous Cyanobacteria: These include oommon filamentouscomponents of eyanobaeterial mats suoh as: Microcoleu,_ chthonoplastes, and various speoies of Lyngbya,Oscillatoria, Trichode_mtum_ and Gloeothece. The exact strategy is still not known but it may include _emporalseparation of the nitrogen fixation and oxygenic photosynthesis stages.

1.4 tWttrogen in _a, be_us meteortt_ and terrestrial fossils

Although Nitrogen is abundant in living organisms, it is severely depleted in meteorites. *_ During his initialinvestigations of Orgueit, Clo_z _'2 reported the detection of an ammonium compound at approximately 0.1% (weight%). He considered the ammonium to be present as a water sohabte ammonkma and eNorine salt flaat he called

• " _ Mason concluded that the Orgueil ammonium salts were probably in the form'ammonium hych'oehlerate . In 1963, ,s

of.NH4Clor~)2S04' Moore%reportedfinding 2,400 ppm nitrogen (0.24% atomic) inOrgueil and 2,900 ppm inthe Alaismeteorite.Gibson ~tal.,47 :analyzed 27 carbonaceous m4i1teorites and found thatearbon~rich meteorites werealso ~hed.muitrogettThe~~d vtU'iatlOllsin tbenitrog¢n levels ()f mdividl.11:tl samples show the heterogeneousdistribution·ofniltogen inOtgueil.

Nitrogen is also severely depleted in ancient fossils. It enters the geolOjlcaI cycle through the enzymaticfixation of atmospheric N~ $11d it is ~onned into ~onil.1ttl.. GaJliell eel a!, .used N~~ Reaction Analysis(NRA) to mvesUg@te the nitrog~. Itnt;l CIltOQn; co~t of biogenic and abiogenic minerals· in Paleo-zoic shales andfound the followiogatomicCiN ratios:

Abiotic Devonian hydrothermal feldspars:Marine bacteriaBiogenic mineralsProterozoic kerogensArchaean kerogens

CIN = UJ3 - U.26CIN "" 2.9 - 14.3CIN= 17 - 25CIN =104 - 167CIN = 200 - 500

these published results areoousistent with the data from the BOS .investigations carried out at theNASAlMSFC AsltobiologyLaboratory ouawidevatiety of recognizable ttrlcrofossilsin clltbonaeeous meteorites, aswell as fossilsoflli'ilobites, fiShandproktuyotlc. fl1ametttsmtenestrialrocks asweUas the data ohtained from EDSstudies of living .1.ID,daniJientbi,)1ogical materials. Table 2 prOViMS th4i1\')N; C/Oand CfB values fot a ttumher ofrepresentative examples. The Electron Energy-Loss Spectroscopy EELS or NRA syst.ems~ are much more sensitive tothe low-Z elements than the Ette1&Y Dl$pefsive X~r$Y .. S~trO$~py(EOO) systems ~ployed in the presentinvestigation, Even~r ideal ~onditions the EOS rarely detects the low~Z element Nit:rogen at levels as below O,Z%0000 ppm), Howevet,since theseelem.ents are lkteetable at level.<; above 5000 ppm (...0.5%) a value of·0.5% wasused to estimate the lower limit for CINandC!S .ratios 'VI'henNitrogenor SuU'urvalues were retumedasO:OO% by theEnergy Dispersive X-ray Speotrotneter software,

SuIfut is II. major biogenic ele~t that cOt1:JP'rises II; stotflgc component fOlt U1llfly bacteria. The strongcovalent disulfide bortdh~ eJillCial to the fulwng, structute, atltt mnc.tion of proteins. Sulfur is lit ttt1nOlt oortstltuent incarbonaceous meteorites. The sulfur content is highest level (5,9010) in theClcarbonaceous meteorites and thecai'bonates :and sulfates inCl~ CM.tooritespto"lid<ls evi~ for aqueous .activity on the parent body. 4J

AlthQugh sulfur lsctiticll.l for life, itta@lye~eedsa~'reeIlt . living organisms. Howevet, thesuffor content of theOrgueil filaments is extremely high (-1O~50% in the fila:m¢nts) but it is much lower in the Orgueil matrix. This }lQintis dramatically illustrated in the 2D X-ray map of F" ZlJ, where the fllaments glow brightly in Sulfur against themeteorite rock OOtrlx lJaekgmurtd,. 1111,';~l~vels .found IlW. the filaments of the OtgueiJ met¢Qnte are signifi~tlyhigher than p~sent in livmg cyanobacteria and other micro\>lal extrettlophiles,

Phosphorus is j},ff}:tcial component of the RuBisCo .euzymeand it isesseutial for the nucleotide adenosinelli'iphosphate (AT.£» - the mt\jOtettetgycUJ;t~cyofc¢lls. However phosphorus is a minor component of the CIm~t:iUls"';\5andispl'esentat~ jOSwe'ight%:t'ottheOrgueil 'l'neteoti1;e.'Phospn01'olJ,ll is l'arelydetected in the Orgueilfi~tslUldit is typj.caUyat very low levels «05%) in. livirtg eYWJobtt¢'tetla, hacteria, arehat::aand diatoms.

Magn~ium is a minor biogenic element in terms oithe amount present in cells, but it is used in enzymes andplays a major role inphou>syttthesi$ .ijS. a COn1POnettt of chtotQphyll. ~ag11leSil.1ttl. is a tllajmr element in the CImeteorites, .where it oomptises ~.6% by wei~t. Much oftbe ma~esium is in water..solnble hydiatedmagn~ium

sulfate (indifferent stages ofnydration) and as hydrated Mg layer-lattice silicates (serpentine or chlorite).44

2. RESULTS

During the past decade, the ElectroSean Environmental Scanning Electron Microseope (ESEM); the FEIQuanta 600 FEG and the Hitachi 8-4100 Field Emission Scanning Electron Microscopes (FESEM) were u,,'led at theNASA Marshall Space Flight Center to produce high resolution images and Energy Dispersive X-ray Spectroscopy(EDS) analysesanO. 2-D X~raymapsof e1ementaloompositions of embedded .microfossils in ancient rocks andcarbonaceous meteorites. Comparative studies with the same in8ti'Umentation was carried oLit on a wide variety of

5

megafossils (Trilobites, Fish, etc.) as well as known fossils of Proterozoic and Archaean cyanobacteria and associatedfIlamentous prokaryotes and environmental samples and pure cultures of living cyanobacteria and axenic cultures oftype strains of novel genera and species of bacteria and archaea. Freshly fractured interior surfaces of many of the CIand CM carbonaceous meteorites have been found to contain many complex and embedded filaments consistent insize, shape, and morphology with known species of cyanobacteria and associated filamentous trichomic prokaryotes.

An ElectroScan ESEM image at 15 keV of a filament in the Murchison CM2 carbonaceous meteorite isshown in Figure l.a. This image is interpreted as the mineralized remains of an emergent hormogonium from amorphotype of cyanobacteria (cf. Nosloc sp.). The EDS spectrum~ Lb) at spot X shows C 41.7%; 0 16.8%; S6.1% and N 0.0% (taken as N<O.5%) gives elemental abundance mtios: C/0=2.5; C/N>82; C/S=6.8. Detectable levelsof Iron and Nickel are observed. The suite of elements is consistent with that observed in the meteorite matrix at pointY providing a clear indication that the filament belongs to the meteorite and it is not a recent biological contaminant

(" ,j I - ••Si Ill5"o II•.~·.\1:: ~~ ••F" It ...... '"" (, 1't It

P 1.1 0

\1 ll-l

"I 11.6 0 •

{'.I 111.'" 1111 •

1l1ll'1ll1l~"llilllll .u 'I.

I<lgurela. Filament with crosswall constrictions in the Murchison CMl meteorite interpreted as emergent hormogonium of speciesof Nostocacean cyanobacteria. b. EDS spectrum at X shows elemental composition of filament similar to Murchison matrix butenriched in Carbon (C=42%; 0=16.8%; S=6%; Ni=O.6%; N<O.S%; CIN=82; C/0=2.S;C/S=6.8).

2.2 Images and EnS ele ental ab eel of the OrgueU ell Meteorite

The vast majority of the embedded filaments in the Orgueil cn carbonaceous meteorite have electrontransparent carbon-rich sheaths enveloping a permineralized interior rich in magnesium and sulfur. This is interpretedas the result a fluid infused with a magnesium sulfate solution infilling the hollow carbonized sheath after death of thefIlamentous prokaryote. It has been known since shortly after the meteorite fell in 1864 that the Orgueil meteorite is amicroregolith breccia that disintegrates immediately when it comes in contact with liquid water. 1

-3 Consquently, it is

suggested that the infilling of the interior of these carbon-rich envelopes must have occurred on the parent body priorto entry into the Earth's atmosphere. Some of the envelopes of the Orgueil fIlaments and sheath-like electron­transparent envelopes have over 80% (atomic) carbon. All of the filaments found have extremely high levels ofmagnesium and sulfur, This result is consistent with a magnesium sulfate rich tluis infilling hollow carbonaceoussheaths of cyanobacteria or filamentous sulfiur bacteria after death. This would require a flow of liquid water andsince the Orgueil CI I meteorite is destroyed by liquid water, this observation is interpreted as providing strongevidence that the perrnineralization of the ftlaments took place on the parent body prior to entry of the meteorite intothe Earth'g atmosphere. F re 2.a. is a Hitachi Field Emission Scanning Electron Microscope (FESEM) image of asmall spiral filament in Orgueil with size and morphology simjlar to known representatives of the modern helicalcyanobacterium Spiro/ina sp. The EDS spectral data from spot X is shown in Fi 2••

Figure 3& is a a JOOOX FESEM image of a very small (-120 /IDl) fragment of the Orgueil meteorite withFig. 3.b. a 2D X-ray elemental map. This small region is densely populated with many different types of embeddedfIlaments and electron transparent sheaths. Several of the filaments have complex morphological features that are wellknown in modern cyanobacteria and other trichomic prokaryotes. The major filaments and sheaths are clearly seen asbright features in the C, 0, Mg and S maps and they appear as dark features in Si, Fe, and Ni maps due to the

6

relatively higher content of these elements in the underlying Orgueil meteorite rock matrix. Filament 1 can be clearlydiscerned in the Nitrogen map and the wrinkled, electron transparent, empty sheath 7 has a relatively high (47%)content of Carbon content. It is one of the only filmnents ever found in the Orgueil meteorite with detectable levels ofboth Nitrogen (1 %) and Phosphorus (0.8%). The irregular longitudinal striations of ftlaments 1 and 1 suggest these aremultiseriate filaments in which multiple parallel oriented trichomes are enclosed within a common homogeneoussheath. Both of these filaments appear to be attached to and physically .embedded in the rock or clay substratum of theOrgueil meteorite matrix and thus they may represent epilithic or cpipclic fonus.

1166

874

583

291

5120

Q""nti'~li.('R.,svll:s (.",- E.2-C1njJ>2 • $m.:.11 :spiral iiJarro('nAna IllS is : Bulk Method: S1arodaFdlessAequ .....d 03-S.-p-2Q04. 10.0 K.V@ O.V/clnnn..1

Elo?foent. ·ri..~ht ~ St". [)(.v. I'"n Atomic S'll k-Ratlo.C 3.26 1.09 \.:>2: 7 e.o om 170 '3.87 095 075 2425 00779

N.. ' 0,00 000 3,00 000 0000

9 11.59 .78 1.26 1335 0.0876I'll' 0.S-3 O.ZZ S.74 0.55 0.0041

51 6.07 117 145 605 00530S 41.~ 1.26 0.61 315m 0.3832

CJ? 2.34 0.79 3.42 1.63 0.0218F. 2\ 05 1. 9 441 1054 01814

NI? 0.('0 0.00 5.1<: 000 0.0000lotal 00.00

? T~s .. .. lelTlffits an sl3l 'sh....11y "'s'9nlf~n\

a ~

Figure 2.a. Morphotypes of Spiru/ina sp.in the Orgueil meteorite with b. EDS spectrum showing elemental abundances ofMagnesium sulfate pennineralized filament at spot X (C 7.6%; N < 0.5%; 024%; S 26%; C/N> 15; CIS = 0.3; C/O = 0.3)

Figure 3. FESEM SED and BSED images and 2-D Elemental X-Ray Maps of Orgueil fragment with many different types ofembedded filaments and empty carbonaceous sheaths. EDS spot data for numbered spots given in Table 2.

The end of Filament t widens slightly (-10 /lm) where it joins the rock matrix and it appears to contain fourtrichomes with diameters -2.5 /lm Itrichome. The larger filament 2 (- 20 /lm dia.) has longitudinal striationssuggestive of -5 trichomes with diameters -4 fllIlItrichome. Faint cross wall constrictions are visible in Filament 2suggesting the internal cells are - 4 JlIll in length and hence roughly isodiametric. The inferred configuration of

, filament 2 is that it consists of an ensheathed trichome bundle of parallel trichomes composed of isodiametric cells of

7

4 fUD diameter as is well known in modem morphotypes of lIDdifferentiated fllamentous Oscilliatorialeancyanobacteria of the genus Microcoleus Desmazieres ex Gomont) (See Castenho1z, Rippka & Herdman4

').

Reproduction within this order occurs by trichome fragmentation and the production of undifferentiated shorttrichome segments (known as hormogonia) by binary fission of the cells in one plane at right angles to the long axis ofthe trichomes. The small multiseriate filament 1 is interpreted as representing a morphotype of the genus TrichocoleusAnagnostidis50, which was separated from the genus Microcoleu3 on the basis of cell size and morphology. Trichomesof species of the genus Trichocoleu3 are typically of 0.5fUD -2.5/lm diameter. EDS spot spectral data on the meteoriterock matrix and several of the numbered ftlaments and sheaths are given in TABLE 2.

Ou.~tft.tt""e- ::u..",tts for C~Jt20.. .$he-4.hAr..fysis: !S... k ........~d· Si.-n=--= olPS~

Atqu~ed 25- ....1-9-200..... , SoD Ke-V ~tG .v/·e-........- ..I

"'omt; •8'~

S2:zl~ 2ec;ooC;~

594cooCOO

MI>LJ.'''­:>.4S1.5;'~.10

3-2;913?2.93~ 43

iUd. t.V".I.SoS0."711.021).000 ......0.3AO .•Xi0::.:.

1 ~so

~'79

23S4

1!l89

0 794

!,,atW. Hla slILAI SI ,l,s

IEI~nt ""·."JIbt ..C. 70.6<1:a 10.59

"'9 '3.97"t., Q.QQSi"'" • '6

S 136::>r .. .') 000'41'" 000

.,..... 1 00.00? TtrwosCl' .lti""""~ ....,., s1..a1:.s4: c~II)' L""lS'9l"'!I1'I~ant.

0000

j

L ~Figure 4.& Orgneil filament with wrinkled de-laminated carbon-rich sheath. b. EDS spectra at spot X on sheath.

Figure 4.a. is a Hitachi FESEM image at 6000X of a complex, curved and polarized filament in Orgueil. Thebasal region of this branched filament is -8 /lm diameter and primary trichome tapers to 3 ji1Jl diameter at the apex.The terminal end of the filament is covered with an electron transparent mucilaginous sheafu that encloses a 0.4 /lmdiameter tenninal hair. The fIlament exlubits both "T" and "Y" branching configurations and the secondary trichomesare much narrower than the primary trichome. The secondary trichome at the lower center of the image forms a "Y"branch and then terminates in rolIDded empty sheaths. Ine Y branching and other features are suggestive ofcyanobacteria of the Order Stigonematales, Geitler. 51 Some modem representatives of the genus Fischerella havebranches that are much narrower than the main filament. This Order includes species that grow in thermal springssuch as MastidocIadopsis Iyengar ct Dcsikachary.52 The Orgueil filament has a large nodule near the base that mayrepresent lateral heterocysts, such as is sometimes seen in the genus MastidocIadopsis. This genus has not beenextensively studied and only two tropical freshwater species (often found attached to stony sUbstrates) have beendescribed. The sheath of the Orgueil filament is wrinkled and laminated, which may be the result of the conditions inwhich fossilization took place. It should be also noted that the modem genus HapaIosiphon also has species withtapered and curved main filaments -8 fUD (e.g., Hapalosiphon welwitschii 5-7.5 J.1Dl). Hapalosiphon hansgtrgi has 6-8/lm diameter main filaments narrowing to about 5 fUD at the apex but none have been reported with such narrowbranches. The EDS speetrum of the sheath (4 X) is exceptionally enriched in carbon (82 % C) and has a C/O ratio of8.9. This is consistent with coal or kerogen hut dramatically different from the samples of living, recently ancient(pleistocene and Holocene) biological material. The EDS spectra for the filament interior (4a¥' Table 2) shows it ispermineralized with magnesium sulfate and bas nitrogen content below the detection limit.

8

2.3 Images and EDS elemental ab ces ofArchaean fiblDM:ntc:tus prokaJ"yota

During this study we also evaluated the CIN, CIS and CIO ratios from fossil filamentous trichomicprokaryotes and cyanobacteria from the Upper Archean (lopian) rocks ofNorthem Karelia. Specimens from the UpperArchaean (lopian) deposits of Northern Karelia were collected by V.A Matrenichev and N.A AlfImova from theInstitute of Geology and Geochronology of Precambrian of the Russian Academy of Sciences and provided by Prof.A Yu. Rozanov, M M AstafJeva, and Y.E. Malakhovskaya of Paleontological Institute (RAS). The samples werecollected from the Northern part of Khisovaar structure (parandovsk-Tikshosersk greenstone belt) which consists ofthick complicated complex of volcanogenic-sedimentary rocks. The upper part of sedimentary complex was found tohave isotopic age of 2706±7 million years and the lower portion 2803 ±35 million years. ~3~4

L b. ~

Figure 5.a Mineralized filaments from carbonaceous shales of the Upper Archaean (lopian) rocks (-2.7 Ga) ofNorthem Karelia. a.Morphotype of oscillatorialean cyanobacteru8 with external nodules on isodiametric filament with b. EDS elemental composition.c. Unusual segmented filament of unknown affinity in Karelian rock. (EDS spectral data for spot X provided in Table 2 as 5cX.)

2.4 Images and EDS ab trilobites, Dfl'o' Mites, and Eoeene fish

Energy Dispersive X-ray Spectroscopy analyses were also carried out to determine elemental compositionsof well known terrestrial fossils such as Cambrian and Ordovician trilobites, Devonian mites and cyanobacterialfJ.laments and Eocene fIsh. . re r6.a is a visible light image of Brachyaspidion microp.~~ This is small well­preserved Middle-Cambrian (-500 Ma) trilobite of Order Ptychopariida was collected by the author in the WheelerSprings Formation, House Range, Millard County, Utah. The Wheeler Shale mudstones are comprised exclusively ofa fine-grained mixed carbonate mud and clay that accumulated below the influence of storm waves. The WheelerShale contains a very rich and diverse biota, including an abundance of benthic trilobites (e.g., Asaphiscus wheeleriand Bollaspidella wellsvillensis and many soft-bodied members of the Burgess Shale fauna. The Wheeler Formationaccumulated in a deep, localized, fault bounded trough known as the House Range embayment on a broad sulfur-rich,carbonate platform.~7 The presence of Burgess fauna and Burgess Shale type preservation indicates an anoxicdeposition in the absence of bioturbation. These are ideal conditions for extensive production of benthic, sulfur­oxidizing anaerobic chemolithoautotrophs, such as Beggiotoa and Thioploca.

These microbial communities could have provided a rich food source for Cambrian metazoans, such as thetrilobites of the Wheeler Shale. Figure 6. is a visible light image of the common Middle Cambrian TrilobitePeronopsis interstricta (Order Agnostida, Family Peronopsidea) from the Wheeler Formation, House Range, Utah.This small agnostid trilobite had no eyes and only two thoracic segments. F' re 6.c is a well-preserved Ordoviciantrilobite (--445 rna) Reacalymene limba from the Ashgill formation of North Wales. This inflated specimen is 27 romlong and has a semicircular cephalon and small, holocbroal eyes.

A low magnification (900X) ESEM image of a fragment of an Orabatid mite with well preservedtrichobotrias from Devonian graphite of the Botogol deposit of East Sayan (South Siberiai8.~9 is shown in Fig. 6.d. Itis now accepted thate these raphites were formed by the conversion of highly carbonic sedimentary carbonate rocks

9

Fig. 6.e. is a 4000X ESEM image of a filament with size and morphology similar to known trichomic filamentouscyanobacteria of the Order Oscillatoriaca:. The spots at which the EDS data shown (fable 2) were obtained aremarked on the fJ1ament Fig.6.f is a visible light image of the small Eocene (~50 Mya) schooling fish (Knightia sp.)commonly found in the laminated sandstone of the Green River Formation of Kemmerer. Wyoming. The EDSspectral data for the marked spots on the trilobites and the spot C on the mineralized bone just beneath the eye socketof the Eocene fish are given in Table 2.

II II

c.

d ~ f.Figure 6. Middle Cambrian (~500 Ma) trilobites a. Brachyaspidion microps and b. Peronopsis interstricta from theWheeler formation, near Swazey, Peak, House Range, Millard County, Utah Co Ordovician trilobite Reacalymenelimba from AshgiJ] fin. (449-443 Ma) ofNorth Wales. d. Image of Devonian Orahatid mite fragment and e. trichomicfllament in the graphites from Botogol, South Siberia. f Eocene (~50 Mya) schooling fish (Knightia sp.) with EDSspectra in Table 2 from Spot C of the bone just beneath the eye.

10

2.4 Images and EDS ele tal abwlublDces of Pie' to"" fil ts fro Vo tok Ice Cores

The Central Antarctic Glacier at Vostok is -3.7 kIn thick ice sheet that overlays the 500 meter deep LakeVostok. Sabit Abyzov et aL 60-62 have pioneered the study of viable microorganisms from the deep ice of the CentralAntarctic ice sheet above Lake Vostok. They have shown that microorganisms can remain alive in a state of deepanabiosis for many thousands of years. Collaborative in-situ studies of Vostok ice and thawed ice cores precipitatedon membrane filters were carried out at MSFC. These studies primarily used the Electroscan Environmental ScanningElectron Microscope (ESEM) to image ice fragments as they were allowed to melt in the instnnnent chamber. Thismade it possible to absolutely establish that the forms that were observed to emerge from the interior of the Vostok icesamples were indigenous and could not be interpreted as recent biological contaminants. Ahundant microorganismswere found in all layers (102 M to 3611 M) of the ice sheet examined Great variations in the composition, density anddistribution of particulates and recognizable microorganisms were observed from one layer to another.

Rhizozol~ni.. al"ta\'u~fok 1002 :\1

(' 22.6%:'\6.5%'<.2%('/'.\ = J.:'CIS >113

Figure 7. ESEM images of Pleistocene microbiota from Vostok ice cores: a. trichomic cyanobacterium from l249Mdepth; b. diatom (cf. Rhizosolenia alata var. gracillima) from 1002 M deep ice layer with Co EDS spectrum at spot X.

Figure 7.a.is an Electroscan ESEM images of a helical coiled cryopreserved filament from the 1249 M layer(- 80,000 yrs) at Vostok. This fIlament has an emergent trichomic structure interpreted as a moprphotype of afilamentous cyanobacteria of the Order Oscillatoriacre. F' Fe 7.". is an ESEM image of a well-preserved diatomfrom 1002 meters depth (age -70,000 yrs). This diatom has been identified as Rhizosolenia alata var. gracillima(Cleve) Gran., which is one of the smaller representatives of the family Rhizosoleniacre. Some of the Rhizosolenia aregigantic. Rhizosolenia. styliformis has been reported with valve diameter up to 100 IUD and lengths exceeding 1500J.lID. The detection of this R. alata in the Vostok ice is interesting, since fue family Rhizosoleniaceae is a marineplanktonic diatom with no known freshwater fonns.63 The EDS spectrum in Fig. 7.e. shows that the nitrogen contentof this -80,000 year old diatom from the Vostok ice core is similar to that of modem living diatoms.

2.5 Images and IDS abundances ofHairffi sue ofPleistocene Mammoth and Pro-d .6 Egyptian Mummy

The FEI Quanta FEG Scanning Electron Microscope was used to obtain high resolution images and conductEDS Elemental analyses of -15,000 year old samples of Wooly Mammoth guard hair, undercoat hair and tissue. ofMammuthus primigeniu3, Blumenbach, 1799. These samples were collected by the author in 1999 during theInternational Beringia Expedition to the Kolyma Lowlands of Northeastcm Siberia. In 1977, an exceptionally well­preserved 40,000 year old frozen carcass of the baby Woolly Mammoth Dima was recovered near Magadan., Siberiafrom the permafrost near a tributary of the Kolyma river.6-4 Figure 8... is an image of a -200 J.lm diameter guard hair.

II

The square spot on this image is the result of beam damage to the hair by exposure to the 10 KeV electron beam as theEDS data for this area shown in F~ 8.• were obtained. Figure S.c. is an image of a small fragment of Mammothtissue with several undercoat hairs still attached The X is linked to the dark square produced by beam damage whenthe EDS data was taken on the mammoth tissue sample and the 5000 year old hair Fig. 8d. from the pre~nastic

Egyptian mummy. All of these ancient hair and tissue samples show strong Nitrogen peaks with CIN and CIS ratiossimilar to living biological materials. Beam damage is the result of heating breaking down the proteins,polysaccharides and other organics. It is very frequently observed when studying modem, Holocene and Pleistocenebiological materials. Beam damage is only rarely seen during investigations of the fllamentous microfossils found inthe carlxmaceous meteorites or in the nrineralized microfossils and macrofossils found in Archaean, Proterozoic, andPhanerozoic (except Pleistocene and Holocene) rocks on Earth.

a. b. c. d.Fi 8.. MamuJhus primigenius guard lair with b. EDS spectra at spot X and Co Mammoth tis ue and undercoat hairs and d.FEI Backscattered electron detector image of pre--d:ynastic Egyptian mummy hair (5,000 Yrs.) showing beam damage at spot Xwhere EDS spectral data shows stroog Nitrogen peak (C 64.3%; N 10.7%; 0 19.6%; S 1.8%; P <.5%; CIN=6; C/S=52; C/0=3.3).

2.6 Images and EDS el mental abundances of modem di toms, b cterl and cyanobacterl

The investigation also included determination of the ratio of biogenic elements in diatoms preserved for almost over150 years on Herbarium sheets at the Henri Van Heurck Museum in Antwerp, Belgium as well as well as livingcyanobacteria and bacteria (Flg. 9). The sample in Flg. 9.a. was collected by Hoffman Bang in 1816 and designatedBangia quadripunctata with an epiphytic filament attached to the diatoms. The EDS data for the filament at spots Xand Y and the diatoms at spot Z are given in Table 2. The 500X FE! Quanta 600 FEG scanning electron microscopeimage of the type series of the diatom is shown in Fig.. 9.b. These small naviculoid diatoms were collected byLenormand in France in 1S34 and mounted on an herbarium sheet and subsequently described by Kutzing65 asSchizonema lenonnandi Kutzing, 1849. These diatoms were observed by the author to emerge from broken ends oftheir gelatinous sheaths and begin swimming after sterile distilled water was added to a well slide containing a smallfragment of the filaments. 66, 67 9.c and d. are Hitachi FESEM images of the living cyanobacteria Lyngbya subti/isand d. a collapsed filament ofOsctl/aloria /ud,grown in pure culture at the NASAINSSTC Astrobiology Laboratory. Figure 9.e. isa FESEM image of a living sample of axenic culture of the type strain Spirochaeta Americana, Hoover 2002 that was isolated fromthe sulfur rich black mud sediments of Mono Lake in California. The EDS spectral data (Fi~ 9.e) at spot X 011 a dump of the tiDyhelical coiled filaments shows a clearly delineated Nitrogen peak: (N = 10.7%, atomic) with C=62.3%; 0 = 20.8% and S = 0.4%(CIN=5.8; C/0=2.7; C/S= 156. Figure 9.f. is a FESEM image of a living sample of axenic culture of type strain of microbialex1remophiles68 Spirochaeta Americana, Hoover 2003 and the EDS spectra showing a clearly delineated Nitrogen peak: is in Fig.9~ The EllS data for the other samples are provided in Table 2

The EDS spectral data shown in Table 2 establish that the diatoms and fllaments, which have been stored indry condition since 1834, have Nitrogen levels and CIN ratios are consistent with living microorganisms. Thesemeasurements on mammoth and mummy hair and tissue and diatoms and their associated gelatinous envelopespreserved dry on herbarium sheets for over a century and a half provide solid data indicating that the loss of nitrogenfrom biological samples occurs over geological rather than historical time scales. Consequently the absence of

12

nitrogen in the fIlamentous microstructures found in freshly fractured interior surfaces of the carbonaceous meteoritesindicates they should be interpreted are indigenous remains rather than recent microbial contaminants.

..("",,t~\ ....... :l[ '$1., l~., ..... .. 7 -.'t ~! "', 1 -,~ 0'8 ~1 31 f) !".:l3-

" lu ObB ":. ,. 0 ~..'J ...~ ,.- ll...... v ...es .. Ie:.; 0 !lJ

'" '3; ... 0- na· 0«· I" 0.,. 1 0'• 071 0" ocm·" o~J 0 .. I.'.' 0_ OD:!..o~. , >C o$<) o 'Sf) o 7'J OO,'!>7P ...0 04e 0'55 0,' Or,,1::1< ltn 035 05. 0.: .. 0001

01 ...· u"" .·'H " 00: fI., (,(,(. r'(fit " "'lO ooסס0

c. I T 0., 071 ~~,. 00111'. 000 C·Y) ~ t:: ·000 O<)lOOTv"l l'-.JOUCJTlw~ ..1iH•....,t ~. ' .. t..:fK.Il)'" .... 4 • ..; .... '

~ [ ~

l'igure 9. Dried herbarium samples from 1816 of epiphytic filamentous forms on the diatom a. Bangia quadripun£laJa and b. thediatom &hizonema lenormandi Kutzing, 1849 marked where EDS data were obtained. c. the living cyanobacteriumLyngbya subtilis and d. a collapsed filament from an axenic culture of Oscillatvria Iud, eo living spiJal filament of thecyanobacterium Arthrosopirs platensis showing beam damage and f. living sample of axenic culture of type strain of microbialextremophiles Spirochaeta Americana, Hoover 2003. and r. (EDS spectral data at spot X shows N = 10.7% (atomic) and S = 0.4%(atomic) and ~ living EDS spectral data for other samples provided in Table 2.

3. BIOGENIC ELEMENT RATIOS

Table 2 provides the compilation of the elemental abundances measured for a number ofthe macrofossils and microfossils from carbonaceous meteorites, Archaean, Proterozoic and

13

Phanerozoic rocks and Pleistocene, Holocene and living biological materials studied during this

investigation.

TABLE 2, Biogenk Elements in Carbonaceous Meteorites, Archaean to Pleistocene Fossils,Holocene and Recent Diatoms and Cyanobacteria

FOILM C O N P big g Si A1 Fe C_o c/N c/s [

5lnrehison Matrix I_Y 6.2 gO 1 1_6 0.3 /._ 2,6 10.8 0.9 6.1 0.i2 o,,, [Orgaeil_'dau;ix_3a8 3.9 69 0.0 0.4 06 41.4 t.=_ 0.2 44.1 0.6 >7 0.t

(_.guoll _.'_lan'lx_BMls_3al2 1.2 6_4 02 <.5 1,1 1.9 1.7 0.0 86.4 0.2 6.0 0.6

_lnrchison_Hormogonl_'_ 41_7 16.8 *:.5 12 7.4 6.1 18.5 0.4 6.4 2.5 >83 @4

OrguellFlt_ment_2_lX 7.6 24.:3 .".5 .-':.5 13A 36.0 6.1 0_6 10.5 0.3 ::-15 0,3Or_eflFilamea_3_l 712 398 08 0.2 13.9 34.8 t.3 '0,2 'l.f '0. t8 >1,1 02

Orguell_t'll_meat_3a2.... 198 17.2 <5 1.0 17.3 41.5 1.8 0_0 1.0 2.8 ::20 0.5 [IJ.,."g 0.2 _" _ 9 [ 0.6 " _ 7.7OrgueR Filament 3_3 22..4 _*__4 t.3 10.9 _.. 0.4 1.7

1 1 [

Orgue|lShe*ath$_7 465 20.0 1.0 0.8 7.4 8_8 12.f. t_4 !.2 [2.3 20 5.3

Orgueil__Filameat_3_9 8.9 35.1 <_5 0.2 t4.2 338 4,0 0,3 3.1 0,3 >18 0,3

Or_eil_ _Sho_th_3_10 54.8 1__,4" <-.._' 0.6 6.t 1-':,__ 6.2 0,6 0.5 4.t >109 a..... 9 0.6 0.5 >91OrgaeiI Sheath 3ali 458 14_8 <'_ 06 8.5 21.5 _ ...

Orgueil Sheath 4_X >7_ 9.2 *: _ - _ _ _............. o 5.9 0.6 i 0.0 0,0 8.9 >156 t3.8

Or_eil_Fihmeut 4,_Y 55.8 11.8 <.5 .r5 11.6 20 0.7 0.0 0.0 ,4.7 , >11i 28

Karelia__Os¢illatoria 5aX -_'__ "_-' ., ,, 2 1.2..... _.4 < _ < _ 0.7 0.0 9,1 0.7 0.8 >44 >&

Kar_lian Filamem SeX 15.6 _68 < g 1.3 0.0 it.2 I 1.6 52,3 0,0 2 3 >31 1.4

Br_ehy_spiaieDEy_X 9 6 [ 44 <5 <_5 2.0 0.0' 24.5 5.6 2.2 4.4 ::-19 >19Peronopsis iuTersn'icta_6bX 9 40 < "_ < _ i .6 0.0 4.5 _ € 4.9 02 >18 > 18

Bologol_Or.qM_i__6ckX 20.4 t5.8 <5 <5 <.5 <.5 26.9 1 t.9 3.5 1.3 :,41 :,41Botegol_Yitame_t_eX 629 3.7 _.5 _::.5 0.0 0.3 15.4 8,8 2.3 t7 >I26 >126Knightia P_le 6fC t g -_ _v 6 .,- _ t _ ._ _ _ i _ 4 g v _ _ 1.6 0.3 >26 9.4 '

Vostok_1249_l_ 7rex , 19,0 66.7 .....a 7 ._ g. _--..,_ <...g 1.1 1.8 0.0 0.3 5.1 >38Voslok 101)2MRhtzo_.%X 33.6 35.t 65 <.5 ' 1.5 '<.5 ' 15,1 ' 5.8 ' 0.9 1.0 5.2 >67

_ammod__gaa_tSaX 704 15.6 11.9 0.0 0_1 t.5 0.2 0,0 0.0 4.5 5.9 46.9 IZ}Lnmmolh tissue. SeX " 59A i 268 4,8 0.1 0.7 0_4 6.3 0.0 0,7 " 2.2 12.4 150

_I_]mmy_tL_U'_SdX 64.4 19.6 10.7. 0.0 0.1 1.8 0,2 0,0 ,0.0 3.3 6.0 ,36181_Baagis.gmX 67.9 21.9 7,1 0.2 0.1 05 1.7 0.0 0,0 3.1 9.6 136

1816..........Ban#a 9aY 62_4 _ 4 9.3 07 0.0 t.0 _ 1 0.2 0.0 " _g 6.7 62.4

l$1__Bangia 9._Z _'; f _ . :' 33.8_;) 4_ 3.4 0_1 0.0 t_8 17,4 05 0.0 0.6 7.9

18.!4Schi__lenormandi_,ghX 50.0 30.4 t0,2 0.0 0.0 <.5 8.5 0.9 0.0 t.6 4.9 100

1834S¢hi__tenorm'andi_gbY 64.9 20.8 7,7 0.0 0.3 ....5 3,1 2,4 0,0 3.1 8,4 130

Lr_.o_suZ, tili_._X 61.5 25,7 9.5 , 1.5 0.0 _: 5 0.4 0.2 ' 0.0 2.4 6.5 122OsciMlolorltt I_d 9_LX 51_9 20_9 4,2 0.1 5.4 2.6 0.4 0.0 0.0 _.5_ lz.__"_ 20

_ _." I 2,5 ,,...itChro_p/r#_plrtte_si._._#eX 62.3 a..0 ,x, 0,0 0.0 <.5 0,0 0.0 0.0 _ _ 125'Sl.,troehaeta am_o'o_a _k'K ' 6_ _ 21.8 10.7 0.8 0.2 ] (_4 08 0,2 0.0 2.7 5.8 156

The ratios of the biogcnic elements CJN; C/S and C/O ratios for the oa,-_mnacamus meteorites mad terrestrialfossils (Archaean to Eocmae) and the Pleistocene and Holocenc hail tisst_ and filaments Woolly Mammoths, pre-dynastic Egyptian mummies and modem and living cyanobacteria, diatoms and other microbial cxl,cmophiles

demonstrate. Although the C/O ratios arc not very helpful, it is clear that C/N and C/S ratios may be effectively usedtodistinguishbetweenindigenousfossilsand recentmicrobiological_ntaminanls.Plotsofthesebiogemc ratiosare

shown in Figure 10.

14

160

140

120

100

80

60

40

20

C/N Ratio• "tV ,,",u.< t M "".03"- O't"I·' M~.lrl,l(

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D2aX Otguetl F,t.umml.3dt_Or9t~1 Flf~"'MTH'nl

0)..2. Or91WotI_FI..~U1UI1t

03~')3_0f9u~II_F,Ic\t'm.lnt

0307 Olguoll_SI_(oUIt-•

.3.....91 Otyu.t:ll FII...tIl~1I1

O~.SO_O'-9m!'JI_ShtJ.lth

3.-.\1 Orqu it Shthnh4aX__ OfgUtJil ShtJillh

04.tY OffJI_UIII Flh.lffh.l."

Sal< Ken haChclllulQrto

QS,;X_Kdu.lld FII~IIIt.:I.1

06ttX e.-o hya!jJ}fdlonEyu

6bK PefOIlOP3iQ. '"tol06dX BOf09OI OrnbiHtI06oX. 8 I~,.,I tAi'l)

D5tC KJuqhh.l B 11

D7~)(_Vo!ll~ 1249 Fll.

C71;X Vostok 1002 Rhlzo

.eaX_Mamntoth_Guard8clC. Mamm()1.ll T, ue

OldX Uummy ~OO~'o

.gaX_18 16B.'llllJla

D8aV_ 181GBongia

90Z_1816Biln~I~1

09bX_'~hi,on

D9bY t834SchlZOn

09cX_Lyngbyo su Iii

IdX Oseln Iud

89 X Anh((} l)lr~1

09tx Spuoc-hil~(a

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CIS Ratio-

f"'"-- ------

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o

20

80

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15

c/o Ratio_1 v Mn. ("h.-.,ou Mr:nrix

0308 Ol'ltlull Mill"C3Il1Z Ornu(.·11 .... l'lIJI)C

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4

2-1---1

8 J----------f' ......----i

6

12---l---------·---I

10

18

14J------------;

16

lilgure 10. Plots of the CIN. CIS and CIO ratios for the fossils and living biological materials described.

A swnmary of the range of these biogenic element ratios is as follows:CIN CIS C/O

Orgueil Filaments: 20 to> 156 0.2 to 13.8 0.3 to 8.9Archaean Filaments (Karelia) 31 to > 34 1.4 to >44 2.3 to 4.4Cambrian and Ordovician Trilobites >18 to >41 >18 to >41 0.1 to 4.4Devonian Microfossils & Eocene Fish >41 to >126 >41 >12 1.3 to 82PleistocenelHolocene Hair 5.1 to 12.4 20 to 15 0.3 to 2.7

Although the FESEM EDS is not extremely sensitive to nitrogen, it is certainly capable of detecting nitrogenat the 0.5% level (5,000 ppm),. Under ideal conditions nitrogen can be detected as low as 0.2% (2,000 ppm) or less.Nitrogen levels as low as 0.2% were detected in the meteorite rock matrix and in a form interpreted as an akinete inthe Orgueil meteorite. However, to be conservative the value 0.5% was used to avoid division by zero to determineminimum elN levels of Table 2. ED studies carned out with the same instrumentation have repeatedly demonstratedthat the abundances of major biogenic elements fOWld in the Orgueil fllaments are distinctly different from that foundin living organisms (bacteria, archaea, and cyanobacteria grown in axenic cultures. in enrichment assemblages. and innatural ecosystems), recently (-190 years or less) dead prokaryotic and eukaryotic microorganisms (cyanobacteria anddiatoms of the Grunow Collection, Henri van Heurck Museum), or in ancient (-32,000 years) cryopreservedPleistocene wood., moss, and bacteria from the Fox Tunnel of Alaska. The EDS analysis indicates that nitrogen is wellabove the level of detectability in all of the living and dead (herbarium. material) cyanobacteria with abundancesranging from 4.6% to 12.6%. Nitrogen was also undetectable in the fossilized cyanobacteria found in the proterozoicphosphorites ofK.hubsughul, Mongolia and in the archaean rocks of Northem. Karelia in Siberia.69

•71

4. CONCLUSIONS

The striking feature about the Archaean filaments and the Cambrian., Ordovician., Devonian and Eocenefossils, as well as the forms interpreted as microfossils of ftIamentous prokaryotes in the Orgueil and Murchison

16

meteorites is the almost universal absence of deL,_table nitrogen even though oarbon is sometimes extremely

abundant. However, the nitrogen levels detected in Iong dead biologieal materials (anoient filamentq from Vostok ice

cores, Woolly Marmaoth hair, and a large number of hair and tissue samples from ancient mummies from Egypt and

Chile) are not notably different from those found in livin 8 and _tly dead eyanobaeteria and other biologieal

materials. The C/S ratios of many of the meteorite filaments, and known terrestrial fossils are also dearly

distinguishable from modern and Pleistocene biological materials

ACKNOWLEDGEMENTS

I am grateftd for the Electron Microscopy _rt providerl by Gregory Jerman and James Coston of the NASA

Marshall Space Flight Center that made this research possible. [ also want to thank Dr. Paul Sipiera and the James M.

DuPont Meteorite Collection of the Planetary Studies Foundation, Chicago, Illinois, and Dr. Marfine Rossignol-Striek

and Dr, Claude Perron and the ?Meteorite Collection of the Mus6e Nationale d'Histoire de Paris for providing the

Orgt.mit meteorite samples. I also _ Dr_ Rosemarie Rippka of the Pasteur Institute (Paris), Academician Georgy

Zavarzin and Dr. Ludmila M. Gerasimenko of the Instilxae of Microbiology, RAS (Moscow) and Dr. Sam van

Landingham for many helpful diseu,_ons about the morphology and characteristics of eyanobaeteria in raml

environments mad axenie eultttres, I thank Dr. Ann St_ Amand of Phyeoteel_ [no, Michigan for environmental samples

of Calothrix and other living eyanobaeteria and the late Dr, Walter van den Bergh of the Hemi van Heurek Museum in

Antwerp, Belgium for herbarium samples of early type algal material. I am grateful to Prof. A. Ytr Rozanov and Dr.

Iv[ M. Astafieva of the Irtsfimte of Paleontology, Russian Academy of Sciences, Moscow for helpful discussions

concerning fossil eyanObacteriz and samples of archaean rectos from Norfl_m Karelia and Academician Erik Galimov

for helpful discussions concerning meteorites and biological fraefionation, t "also acknowledge the funding support

provided by the NASA/MSFC Center Director's Discxefionary Fund:, the NASA Astrobiology Institute and the

NASA/JSC Center for Biomarkers in AstrorrmterialS.. which has made this rese_eh effort possl_ole.

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